Alternative nucleotide flows in sequencing-by-synthesis methods

ABSTRACT

A method of sequencing a polynucleotide strand can include providing the polynucleotide strand with a primer annealed thereto and a polymerase operably bound to the polynucleotide strand; successively exposing the polynucleotide strand to the flow of four different dNTPs according to a first predetermined ordering; and successively exposing the polynucleotide strand to the flow of four different dNTPs according to a second predetermined ordering, wherein the second predetermined ordering is different from the first predetermined ordering.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/689,252, filed on 29 Nov. 2012 (currently pending), which is acontinuation of U.S. application Ser. No. 13/157,865, filed on 10 Jun.2011 (now abandoned), which claims the benefit of U.S. ProvisionalApplication No. 61/354,173 filed on 11 Jun. 2010 (now expired), whichare all incorporated by reference herein.

TECHNICAL FIELD

The present teachings relate to nucleic acid sequencing, and moreparticularly, to the correction of errors that can arise insequencing-by-synthesis techniques.

BACKGROUND

Several next-generation DNA sequencing approaches, often referred to as“sequencing-by-synthesis” approaches, use repeated cycles of primerextension with a DNA polymerase to generate a sequence of signalscontaining nucleotide sequence information of populations of templatemolecules. See, e.g., Hert et al, ELECTROPHORESIS, 29:4618-4626 (2008);Metzker, NATURE REVIEWS GENETICS, 11:31-46 (2010); Droege et al, J.BIOTECHNOLOGY, 136:3-10 (2008). A common problem in these approaches isthe “dephasing” of primer extensions because of the accumulation of thecycle-to-cycle effects of incomplete extension and/or inappropriateextensions (“carry forward”), which lead to significant reductions inthe signal-to-noise ratio as sequencing progresses. See, e.g., Ronaghi,GENOME RESEARCH, 11:3-11 (2001); Leamon et al, CHEMICAL REVIEWS,107:3367-3376 (2007); Chen et al, International Patent Publication WO2007/098049. Currently, such inefficiencies are dealt with by signalprocessing software, such as those described in Leamon et al. (citedabove) and Chen et al. (cited above). But as longer read lengths andalternative detection schemes are sought, such as schemes for label-freeextension detection (see, e.g., Rothberg et al, U.S. Patent Publication2009/0127589), alternative methods for addressing signal loss fromincomplete extension or carry forward errors would be highly desirable.

SUMMARY

In various embodiments, the present teachings apply tosequencing-by-synthesis techniques to sequence a template polynucleotidestrand. To addresses the problem of incomplete extension (IE) and/orcarry forward (CF) errors that can occur in sequencing-by-synthesisreactions, an alternative flow ordering of the nucleotides may be used.In various embodiments, alternative flow ordering may reduce and/orcorrect the loss of phasic synchrony in the population of templatepolynucleotide strands that result from IE and/or CF errors.

In one embodiment, the present teachings provide a method of sequencinga polynucleotide strand, comprising: (a) providing the polynucleotidestrand with a primer annealed thereto and a polymerase operably bound tothe polynucleotide strand; and (b) successively exposing thepolynucleotide strand to the flow of four different dNTPs according to apredetermined ordering, wherein the predetermined ordering comprises analternate ordering which is not a continuous repeat of an ordering ofthe four different dNTPs.

In another embodiment, the present teachings provide an apparatus forsequencing a polynucleotide strand, comprising: (a) a flow chamber forreceiving flows of different dNTP reagents; (b) multiple reservoirs thateach contain a different dNTP reagent; (c) flow paths from each of thereservoirs to the flow chamber; and (d) a fluidics controller thatcontrols the flow from the reservoirs to the flow chamber, wherein thefluidics controller is programmed to successively provide flow from themultiple reservoirs to the flow chamber according to a predeterminedordering, wherein the predetermined ordering comprises an alternateordering which is not a continuous repeat of an ordering of the fourdifferent dNTP reagent flows.

In another embodiment, the present teachings provide a method ofperforming template-based extension of a primer, comprising: providingat least one template polynucleotide strand having a primer andpolymerase operably bound thereto; and successively exposing thetemplate polynucleotide strand to a plurality of each kind of flow suchthat (a) a flow of one kind is always followed by a flow of a differentkind; and (b) at least one flow of each kind is followed by a flow ofthe same kind after a single intervening flow of a different kind.

In another embodiment, the present teachings provide a method ofdetermining the sequence of a template polynucleotide strand bytemplate-based extension of a primer, comprising: (a) delivering a knownnucleoside triphosphate precursor to a template-based primer extensionreaction of a polynucleotide strand, the known nucleoside triphosphateprecursor being delivered according to a predetermined ordering of dNTPflows; (b) detecting incorporation of the known nucleoside triphosphatewhenever its complement is present in the template polynucleotide strandadjacent to the primer; and (c) repeating steps (a) and (b) until thesequence of the template polynucleotide strand is determined; whereinthe predetermined ordering of dNTP flows is defined by (i) a flow of onekind is always followed by a flow of a different kind; and (ii) at leastone flow of each kind is followed by a flow of the same kind after asingle intervening flow of a different kind.

In another embodiment, the present teachings provide a method forsequencing a template polynucleotide strand comprising: (a) disposing aplurality of template polynucleotide strands into a plurality ofreaction chambers, each reaction chamber comprising a templatepolynucleotide strand having a sequencing primer hybridized thereto anda polymerase operably bound thereto; (b) introducing a known nucleosidetriphosphates into each reaction chamber according to a predeterminedordering of dNTP flows; (c) detecting sequential incorporation at the 3′end of the sequencing primer of one or more nucleoside triphosphates ifthe known nucleoside triphosphate is complementary to correspondingnucleotides in the template nucleic acid; (d) washing awayunincorporated nucleoside triphosphates from the reaction chamber; and(e) repeating steps (b) through (d) until the polynucleotide strand issequenced; wherein the predetermined ordering of dNTP flows is definedby (i) a flow of one kind is always followed by a flow of a differentkind; and (iii) at least one flow of each kind is followed by a flow ofthe same kind after a single intervening flow of a different kind.

In another embodiment, the present teachings provide a method ofsequencing a polynucleotide strand, comprising: providing thepolynucleotide strand with a primer annealed thereto and a polymeraseoperably bound to the polynucleotide strand; successively exposing thepolynucleotide strand to the flow of four different dNTPs according to afirst predetermined ordering; and successively exposing thepolynucleotide strand to the flow of four different dNTPs according to asecond predetermined ordering, wherein the second predetermined orderingis different from the first predetermined ordering.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows an exemplary sequencing-by-synthesis process.

FIG. 2 shows examples of IE and CF errors that may occur duringsequencing. FIG. 2 discloses SEQ ID NO: 1.

FIGS. 3A and 3B show an exemplary alternate flow ordering that may beused to improve phasic synchrony in a population of templatepolynucleotide strands. FIGS. 3A and 3B disclose SEQ ID NO: 1.

FIGS. 4A and 4B show an exemplary alternate flow ordering that may beused to improve phasic synchrony in a population of templatepolynucleotide strands. FIGS. 4A and 4B disclose SEQ ID NO: 2.

FIG. 5 is a diagram showing a sequencing apparatus according to anembodiment of the present teachings.

FIG. 6 shows a close-up, cross-sectional view of a flow cell accordingto an embodiment of the present teachings.

FIG. 7 shows sample ionograms from a sequencing simulation of a testfragment.

DETAILED DESCRIPTION

The practice of the present teachings may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, molecular biology (including recombinant techniques), cellbiology, and biochemistry. Such conventional techniques include, but arenot limited to, preparation of synthetic polynucleotides, polymerizationtechniques, chemical and physical analysis of polymer particles, nucleicacid sequencing and analysis, and the like. Specific illustrations ofsuitable techniques are given in the examples below. However, otherequivalent conventional procedures can also be used. Such conventionaltechniques and descriptions can be found in standard laboratory manualssuch as

GENOME ANALYSIS: A LABORATORY MANUAL SERIES (vols. I-IV); PCR PRIMER: ALABORATORY MANUAL;MOLECULAR CLONING: A LABORATORY MANUAL (all from Cold Spring HarborLaboratory Press); Hermanson, BIOCONJUGATE TECHNIQUES, 2nd ed. (AcademicPress, 2008); and the like.

Sequencing-by-Synthesis

The present teachings apply sequencing-by-synthesis techniques tosequence a template polynucleotide strand. In general,sequencing-by-synthesis (SBS) may refer to methods for determining thenucleotide sequence of a target polynucleotide by a polymerase extensionreaction. In various embodiments, the process sequences one or moretemplate polynucleotide strands, which may be provided in any suitablemanner. In some embodiments, the template strands may be coupled to orassociated with a support, such as a microparticle, bead, or the like,and are loaded into reaction chambers. In other embodiments, thetemplate polynucleotide strands may be associated with a substratesurface or present in a liquid phase with or without being coupled to asupport. For example, templates may be prepared as described in U.S.Pat. No. 7,323,305, which is incorporated by reference.

During a typical sequencing reaction, a primer is annealed to thetemplate polynucleotide strand to form a primer-template duplex, and apolymerase is operably bound to the primer-template duplex so that it iscapable of incorporating a nucleotide onto the 3′ end of the primer. Asused herein, “operably bound” may refer to the primer being annealed toa template strand so that the primer's 3′ end may be extended by apolymerase and that a polymerase is bound to the primer-template duplex,or in close proximity thereof, so that extension can take place whendNTPs are flowed. The primer-template-polymerase complex is subjected torepeated exposures of different nucleotides. If a nucleotide(s) isincorporated, then the signal resulting from the incorporation reactionis detected. A wash step may be performed to remove unincorporatednucleotides prior to the next nucleotide exposure. After repeated cyclesof nucleotide addition, primer extension, and signal acquisition, thenucleotide sequence of the template strand may be determined.

The present teachings may use any of a variety of sequencing techniquesand is particularly suitable for sequencing-by-synthesis techniques.Examples of such techniques are described in the literature, includingthe following, which are incorporated by reference: Rothberg et al, U.S.Patent Publication 2009/0026082; Anderson et al, SENSORS AND ACTUATORS BCHEM., 129:79-86 (2008); Pourmand et al, PROC. NAT1. ACAD. SCI.,103:6466-6470 (2006). Variants of sequencing-by-synthesis techniquesinclude methods where the nucleotides are modified to be reversibleterminators (sometimes referred to as cyclic reversible termination(CRT) methods, as described in Metzker (cited above)) and methods wherethe nucleotides are unmodified (sometimes referred to as cyclic singlebase delivery (CSD) methods). The incorporation reaction generates orresults in a product or constituent with a property capable of beingmonitored and used to detect the incorporation event. Non-limitingexamples of such properties that may be associated with incorporationreactions include changes in magnitude (e.g., heat) or concentration(e.g., pyrophosphate and/or hydrogen ions), and signal (e.g.,fluorescence, chemiluminescence, light generation). In the variousapproaches, the amount of the detected product or constituent may bemonotonically related to the number of nucleotides incorporated.Non-limiting examples of suitable sequencing chemistries include thatused on the Genome Analyzer/HiSeq/MiSeq platforms (Illumina, Inc.; See,e.g., Balasubramanian, U.S. Pat. No. 6,833,246 and No. 5,750,341); thoseapplying pyrosequencing-based sequencing methods such as that used byRoche/454 Technologies on the GS FLX, GS FLX Titanium, and GS Juniorplatforms (see, e.g., Ronaghi et al, SCIENCE, 281: 363 (1998); andMaguiles et al (cited above)); and those by Life Technologies/IonTorrent in the PGM system (see, e.g., US 2010/0137143 and US2009/0026082).

In an exemplary conventional SBS method, the four nucleotides aresequentially and repeatedly delivered (flowed) in the same order. Forexample, the first nucleotide delivered may be dATP, then dCTP, thendGTP, then dTTP (or a permutation thereof), after which this sequence isrepeated. Such deliveries of nucleotides to a reaction vessel or chambermay be referred to as “flows” of nucleotide triphosphates (or dNTPs).For convenience, a flow of dATP will sometimes be referred to as “a flowof A” or “an A flow,” and a sequence of flows may be represented as asequence of letters, such as “ATGT” indicating “a flow of dATP, followedby a flow of dTTP, followed by a flow of dGTP, followed by a flow ofdTTP.” In each flow step of the cycle, the polymerase may generallyextend the primer by incorporating the flowed dNTP where the next basein the template strand is the complement of the flowed dNTP. Thus, ifthere is one complementary base, then one base or dNTP incorporation isexpected; if two complementary bases, then two incorporations areexpected; if three complementary bases, then three incorporations areexpected, and so on.

The present teachings may use any of various techniques for detectingthe nucleotide incorporation(s). For example, somesequencing-by-synthesis techniques provide for the detection ofpyrophosphate (PPi) released by the incorporation reaction (see, e.g.,U.S. Pat. Nos. 6,210,891; 6,258,568; and 6,828,100). In another example,some sequencing-by-synthesis techniques may detect labels associatedwith the nucleotides, such as mass tags, fluorescent, and/orchemiluminescent labels. Where detectable labels are used, aninactivation step may be included in the workflow (e.g., by chemicalcleavage or photobleaching) prior to the next cycle of synthesis anddetection.

In certain embodiments, the present teachings may use a pH-based methodof detecting nucleotide incorporation(s). Such an approach may detecthydrogen ions released from the polymerase-catalyzed extension reactionsin the absence of a specific label or tag. The hydrogen ions released bya population of template strands undergoing the base incorporation(s)will change the local pH of the reaction chamber, which can be detected.Thus, in pH-based methods for DNA sequencing, base incorporations aredetermined by measuring these hydrogen ions that are generated.Additional details of pH-based sequence detection systems and methodsmay be found in commonly-assigned U.S. Patent Application PublicationNo. 2009/0127589 and No. 2009/0026082, which are incorporated byreference. While the examples below are discussed in connection withpH-based sequence detection, it will be appreciated that the presentteachings may be readily adapted to other sequencing approaches such asthe exemplary technologies mentioned above including pyro-sequencing.Such approaches can likewise benefit from the phase correction, signalenhancement, improved accuracy and noise reduction features of thealternative nucleotide flows approaches described herein and areunderstood to be within the scope of the present teachings.

It will be appreciated that in connection with pH-based detectionmethods, the production of hydrogen ions may be monotonically related tothe number of contiguous complementary bases in the template strands (aswell as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereis a number of contiguous identical complementary bases in the template(i.e., a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, is generallyproportional to the number of contiguous identical complementary bases.(The corresponding output signals may sometimes be referred to as“1-mer”, “2-mer”, “3-mer” output signals, and so on, based on theexpected number of repetitive bases). Where the next base in thetemplate is not complementary to the flowed dNTP, generally noincorporation occurs and there is no substantial release of hydrogenions (in which case, the output signal is sometimes referred to as a“0-mer” output signal).

In each wash step of the cycle, a wash solution (typically having apredetermined pH) is used to remove residual dNTP of the previous stepin order to prevent misincorporations in later cycles. Usually, the fourdifferent kinds of dNTP are flowed sequentially to the reactionchambers, so that each reaction is exposed to one of the four differentdNTPs for a given flow, such as in the following sequence: dATP, dCTP,dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and so on, with the exposure,incorporation, and detection steps followed by a wash step. An exampleof this process is illustrated in FIG. 1, which shows a template strand682 attached to a particle 680. Primer 684 is annealed to templatestrand 682 at its primer binding site 681. A DNA polymerase 686 isoperably bound to the template-primer duplex. Template strand 682 hasthe sequence 685, which is awaiting complementary base incorporation.Upon the flow of dNTP (shown as dATP), polymerase 686 incorporates anucleotide since “T” is the next nucleotide in template strand 682(because the “T” base is complementary to the flowed dATP nucleotide).Wash step 690 follows, after which the next dNTP (dCTP) is flowed 692.Optionally, after each step of flowing a dNTP, the reaction chambers maybe treated with a dNTP-destroying agent (such as apyrase) to eliminateany residual dNTPs remaining in the chamber, which can cause spuriousextensions in subsequent cycles.

FIG. 2 shows an example of IE and CF errors. FIG. 2 shows three DNAduplexes. For each duplex, the bottom row of boxes represents thetemplate polynucleotide strand 32 and the top row of boxes representsthe complementary extension strand 30 being extended by the polymerase.The extension strand includes the primer portion, as indicated by thebar. In this figure, the ()-filled boxes indicate the incorporation ofcomplementary nucleotides.

The top DNA duplex (labeled “in-phase”) represents members of thepopulation that are in the correct phase, i.e., in-phase. The middle DNAduplex (labeled “IE”) represents a portion of the population that hasexperienced an exemplary omission at the C nucleotide, i.e., anincomplete extension error causing dephasing of the population. Thebottom DNA duplex (labeled “CF”) represents a portion of the populationthat has experienced an exemplary erroneous incorporation at the Gnucleotide, i.e., a carry forward error causing dephasing of thepopulation.

Alternate Flow Orderings

The present teachings address the problem of incomplete extension (IE)and/or carry forward (CF) occurrences in sequencing reactions by usingan alternative (non-sequential) ordering for delivering nucleotides.This alternative ordering may reduce and/or correct the loss of phasicsynchrony in the population of template polynucleotide strands thatresult from IE and/or CF occurrences. As used herein, an “alternativeordering” of dNTP flows means that the ordering is not a continuousrepeat of an ordering of the four different dNTPs. In other words, in analternate flow ordering, the dNTPs are flowed in an order that is not acontiguous, sequential repetition of the same 4-member units, each4-member unit being a sequence of the four different dNTPs. Thisalternate flow ordering represents at least some portion of thesequencing run.

The alternate flow ordering may be reflected in the overallpredetermined flow ordering in any of various ways. In some embodiments,the alternate flow ordering constitutes one or more parts of the overallpredetermined ordering, with one or more other parts of the overallpredetermined ordering using a conventional flow ordering (i.e., not analternate flow ordering). For example, the alternate flow ordering maybe used intermittently with a conventional flow ordering. In someembodiments, the overall predetermined ordering consists of only thealternate flow ordering throughout the sequencing run. In someembodiments, the method may be implemented with real-time detection ofCF and/or IE errors and applying the alternate flow ordering in responseto the error detection. For example, the alternate flow ordering may beused when the CF and/or IE errors reach a certain threshold level. Insome embodiments, the alternate flow orderings may be used according tothe position in the sequence read. For example, the alternate flowordering may be used after a certain read length of the sequence or usedmore frequently at later stages of the sequence read. This may be usefulin instances where the CF and/or IE errors increase at later stages ofthe read or in longer reads.

In some cases, the overall predetermined ordering comprises a firstpredetermined ordering for the flow of four different dNTPs and a secondpredetermined ordering for the flow of the four different dNTPs, withthe second predetermined ordering being different from the firstpredetermined ordering. For example, the first predetermined orderingmay be a conventional flow ordering and the second predeterminedordering may be an alternate flow order.

In certain embodiments, the present teachings may be directed to anysequencing method (including SBS methods) where delivery of dNTPs to areaction is not a continuous repetition of the same initial ordering offlows of the four dNTPs, such as: ACGT-ACGT-ACGT- . . . and so on. Suchan initial ordering of dNTP flows may be any permutation of ACGT, suchas ACTG, TGCA, and so on. The alternate flow ordering may be implementedin a variety of different ways. In certain embodiments of the presentteachings, the dNTPs are delivered in a predetermined ordering thatcomprises an alternate ordering where (a) a flow of one kind is alwaysfollowed by a flow of a different kind; and (b) at least one flow ofeach kind is followed by a flow of the same kind after a singleintervening flow of a different kind. In some cases, the number of flowsof each kind in the alternative ordering is the same.

If “N” is used to represent the flow of any one of dATP, dCTP, dGTP, ordTTP, then in one example, the predetermined ordering of dNTP flows caninclude the following subsequence: N-W-N for each dNTP N, where W is anydNTP not N. In another example, the ordering of dNTP flows can includean alternate ordering with the following subsequence: N-W-N-Z for eachdNTP N, where W is any dNTP not N, and Z is any dNTP that is neither Nnor W. Flow orderings of the present teachings may have a variety oflengths, which are the total number of flows making up a predeterminedordering. In some cases, the lengths of the flow orderings may beprovided in subsets comprising a multiple of defined base flows. Forexample, the length of the flow orderings in the present teachings maybe any multiple of four, eight, or other multiples. An exemplary 8-flowordering of the present teachings is AT-AC-GC-GT, where the “GC-G”subsequence (representing a N-W-N sequence) is present. Note also thatthere is a “T-AT” subsequence (representing a N-W-N sequence) when the8-flow ordering is contiguously repeated. In certain embodiments,alternate flow orderings of the present teachings have a length selectedfrom the group consisting of 8, 12, 16, 20, 24, 28 and 32. However, itwill be appreciated that other flow ordering lengths may be used.Exemplary alternate flow orderings of 12 flows are TCT-AGA-CTC-GAG andACA-CGC-GTG-TAT. An exemplary alternate flow ordering of 20 flows isTACAT-ACGCA-CGTGC-GTATG, which may be repeated one or more times (inpart or whole) to sequence a desired template length.

In another embodiment, the alternate flow ordering includes a first dNTPflow, a second dNTP flow, a third dNTP flow, and a fourth dNTP flow,with each flow being a different dNTP; wherein the fourth dNTP flow doesnot occur until at least one of the first, second, or third dNTP flowsare repeated at least once. For example, for the exemplary 8-flowordering of AT-AC-GC-GT given above, the G nucleotide is not floweduntil each of A, T, and C are flowed, with A being flowed twice.Likewise, for the exemplary 12-flow ordering of ACA-CGC-GTG-TAT, the Tnucleotide is not flowed until each of A, C, and G are flowed (with Abeing flowed twice, C being flowed three times, and G being flowedtwice). In some cases, the number of flows for each of the fourdifferent dNTPs in the alternate flow ordering is the same. For example,for the exemplary 8-flow ordering of AT-AC-GC-GT given above, each ofthe four nucleotides are flowed twice. Likewise, for the exemplary12-flow ordering of TCT-AGA-CTC-GAG and ACA-CGC-GTG-TAT given above,each of the four nucleotides are flowed three times.

In various embodiments, the inclusion or removal of any flow of aselected nucleotide from a series of flows of a sequencing run may beused to impart an alternative flow ordering according to the presentteachings. The number and/or type of flow may, for example, be as few asa single added base flow over the course of the sequencing run (orremoval of a selected base flow). As described above, imparting anon-sequential four base flow ordering (e.g., not strictly GATC, GATC, .. . over the entire sequencing run) may provide for improved sequencingquality and/or signal detection by reducing IE and/or CF effects.

The flow of dNTPs can be provided in any suitable manner, includingdelivery by pipettes, or through tubes or passages connected to a flowchamber. The duration, concentration, and/or other flow parameters maybe the same or different for each dNTP flow. Likewise, the duration,composition, and/or concentration for each wash flow may be the same ordifferent.

FIGS. 3A and 3B show an example of how the alternate flow ordering ofthe present teachings can be used to improve phasic synchrony. FIG. 3Ashows a partial view of two exemplary duplexes A and B, each with apolynucleotide template strand 36 and an extension strand 34. This viewmay represent part of a population of template strands that are beingsubjected to a series of flow cycles during a sequencing run. For eachduplex, the bottom row of boxes represents the template strand 36 andthe top row of boxes represents the complementary extension strand 34hybridized to the template strand and being extended by the polymerase.In the figure, the ()-filled boxes indicate the incorporation ofcomplementary nucleotides in a growing extension strand.

In this example, both templates A and B are represented as havingalready undergone n cycles of a representative conventional, repeatedATGC flow ordering pattern, i.e., ATGC-ATGC-ATGC- . . . . FIG. 3Adepicts only a representative portion of the template/extension strandduplex for cycles n through n+3. During cycle n, the template strand Bis exemplified with an omitted incorporation at the nucleotide base A(marked by the “x”). This may reflect an incomplete extension error intemplate strand B, such that the extension strand is out-of-phase withthat of the template strand A. FIG. 3A shows the extension strandcontinuing to be extended through further cycles n+1, n+2, and n+3 ofthe repeating ATGC flow ordering. As seen here, after multiple furthercycles, the extension strand on template strand B (with the IE error)continues to lag behind the in-phase template strand A. Thisrepresentation of an IE occurrence is only one example and it will beappreciated that many different IE occurrences are possible and can beintroduced at any point during a sequencing run. Moreover, differenttemplate strands may experience different IE occurrences and a singletemplate strand may have multiple IE occurrences at different positions.

FIG. 3B shows the same pair of template strands A and B as in FIG. 3A,also with the same IE error at the location marked “x”. Also, as in FIG.3A, the templates strands have been subjected to n cycles of theconventional repeating ATGC flow ordering. However, from cycles n+1 ton+3, the flow ordering is changed to the alternate flow ordering ofAGA-TCT-GAG-CTC. As seen here, after the third cycle of the alternateflow ordering, template strand B (with the IE error) has beenresynchronized to the in-phase template strand A.

FIGS. 4A and 4B show another example of how the alternate flow orderingof the present teachings can be used to improve phasic synchrony in thepopulation of template polynucleotide strands. FIG. 4A shows a partialview of two exemplary duplexes X and Y, each with a polynucleotidetemplate strand 42 and an extension strand 40. For each duplex, thebottom row of boxes represents the template strand 42 and the top row ofboxes represents the complementary extension strand 40 being extended bythe polymerase. In this figure, the ()-filled boxes indicate theincorporation of complementary nucleotides.

In this example, both templates X and Y have already undergone n cyclesof a conventional, repeated ATGC flow ordering pattern, i.e.,ATGC-ATGC-ATGC- . . . . FIG. 4A depicts a representative portion of thetemplate/extension strand duplex for cycles n through n+3. During cyclen, the template strand Y experiences an erroneous additionalincorporation at the nucleotide base C. This may reflect a carry forwarderror in template strand Y such that the extension strand isout-of-phase with that of the template strand X. FIG. 4A shows theextension strand continuing to be extended through further cycles n+1,n+2, and n+3 of the repeating ATGC flow ordering. As seen here, aftermultiple further cycles, the extension strand on template strand Y (withthe CF error) continues to be ahead of the in-phase template strand X.This representation of a CF occurrence is only one example and it willbe appreciated that many different CF occurrences are possible and canbe introduced at any point during a sequencing run. Moreover, differenttemplate strands may experience different CF occurrences and a singletemplate strand may have multiple CF occurrences at different positions.

FIG. 4B shows the same pair of template strands X and Y as in FIG. 4A,also with the same CF error in template strand Y. Also, as in FIG. 4A,the templates strands have been subjected to n cycles of theconventional repeating ATGC flow pattern. However, from cycles n+1 ton+3, the flow pattern is changed to the alternate flow ordering ofAGA-TCT-GAG-CTC. As seen here, after a single cycle of the alternateflow ordering, template strand Y (with the CF error) has beenresynchronized to the in-phase template strand X.

It will be appreciated that achieving or improving phasic synchronydesirably enhances the ability to identify nucleotide incorporations andcorrectly ascertain the sequence of templates undergoing analysis. Inmany sequencing applications, dephasing issues may be relatively smallearly in the sequencing run; however, their effects may accumulate asthe sequencing progresses and result in degraded sequencing quality whenlonger templates are used. From a practical perspective, it will beappreciated that the corrective effect of the alternate flow orderingwill desirably enhance the base calling abilities of a sequencinginstrument by reducing or eliminating spurious signals associated without-of-phase templates.

In various embodiments, alternate nucleotide flows can be includedwithin or in connection with a series of sequencing flows as a mechanismby which to counteract the accumulated dephasing of templates. Suchalternate flows may therefore be used in some embodiments not tocompletely remove or alleviate dephasing, but rather as a mechanism tobalance or reduce accumulated dephasing effects while at the same timemaintaining an efficient or desirable number of flows to achieve aselected/expected throughout (e.g., the flows used to sequence arespective template length). Use of the present teachings for sequencingmay result in a reduction or correction of CF and/or IE effects,improvement in phasic synchrony, increased signal-to-noise ratio, and/orimproved base calling accuracy.

FIG. 7 shows two ionograms from a simulation of a pH-based sequencingrun for a particular test fragment using 1% as the error rate parametersfor both CF and IE errors. The ionogram in the upper panel shows thesignal peaks acquired using a conventional TACG flow order. The ionogramin the lower panel shows the signal peaks acquired using an alternateflow ordering of TACATACGCACGTGCGTATG. The labeled arrows point to twoexample incorporation signals at the same sequence position on the testfragment. The “4-mer” arrows point to the signals from the same 4-merhomopolymer subsequence of the test fragment. The “2-mer” arrows pointto the signals from the same 2-mer homopolymer subsequence of the testfragment. As seen here, the ionogram in the lower panel (using thealternate flow ordering) has signals that more clearly define thebase-calling classifications as being 4-mer and 2-mer, as well thebase-calling classifications for other signals in the test fragment.

Sequencing Instrumentation

Instruments for delivering reagents for multistep sequencing processesare known, and typically comprise reservoirs for reagents, one or morereaction chambers or areas, and fluidics under computer control forselecting and delivering the various reagents including dNTPs to the oneor more reaction chambers or areas. Exemplary instrument systems forcarrying out massively parallel SBS reactions with electronic detectionare disclosed in Rothberg et al, U.S. Patent Publication No.2009/0127589 and No. 2009/0026082; and Rothberg et al, U.K. PatentApplication GB2461127. Likewise, conventional fluorescence-based SBSsequencing instrumentation are disclosed in Rothberg et al, U.S. Pat.No. 7,211,390; No. 7,244,559; and No. 7,264,929. In fluorescence-basedSBS sequencing instrumentation, the release of inorganic pyrophosphatefrom an incorporation reaction initiates an enzyme cascade that resultsin light emission, which is then detected by the instrument. Thealternate flow orderings of the present teachings can be used with theseand other sequencing methods and systems.

The present teachings also provide an apparatus for sequencing templatepolynucleotide strands according to the method of the present teachings.A particular example of an apparatus of the present teachings is shownin FIG. 5. The apparatus of FIG. 5 is configured for pH-based sequencingand includes multiple reservoirs for containing reagents 1 through K(114). These reagents contain the dNTPs to be flowed for the SBSprocess. The reagents 114 are flowed through fluid passages 130 andthrough a valve block 116 that controls the flow of the reagents to flowchamber 105 (also referred to herein as a reaction chamber) via fluidpassage 109. The apparatus includes a reservoir 110 for containing awash solution that is used to wash away the dNTP reagent of the previousstep. Reagents are discarded through waste passage 104 into a wastecontainer 106 after exiting the flow chamber 105.

The apparatus also includes a fluidics controller 118, which mayprogrammed to control the flow from the multiple reagent reservoirs tothe flow chamber according to a predetermined ordering that comprises analternate flow ordering, as described above. For this purpose, fluidicscontroller 118 may be programmed to cause the flow of reagents 114 fromthe reagents reservoir and operate the valves 112 and 116. The fluidicscontroller may use any conventional instrument control software, such asLabView (National Instruments, Austin, TX). The reagents may be driventhrough the fluid pathways 130, valves, and flow cell by anyconventional mechanism such as pumps or gas pressure.

The apparatus also has a valve 112 for controlling the flow of washsolution into passage 109. When valve 112 is closed, the flow of washsolution is stopped, but there is still uninterrupted fluid andelectrical communication between reference electrode 108, passage 109,and sensor array 100. Some of the reagent flowing through passage 109may diffuse into passage 111, but the distance between referenceelectrode 108 and the junction between passages 109 and 111 is selectedso that little or no amount of the reagents flowing in common passage109 reach reference electrode 108. This configuration has the advantageof ensuring that reference electrode 108 is in contact with only asingle fluid or reagent throughout an entire multi-step reactionprocess. Reference electrode 108 may be constructed in any suitablefashion. In this particular embodiment, reference electrode 108 is atube made of a conductive material which forms part of passage 111.Although FIG. 5 shows the reference electrode 108 as a cylinder that isconcentric with the flow path (to represent the preferred configurationin which a tube of conductive material encloses part of a flow path),other embodiments may use any suitable configuration for a referenceelectrode in a flow path.

As shown in FIG. 5, flow chamber 105 is loaded with a flow cell thatincludes an inlet 102, an outlet 103, and a microwell array 107 which isoperationally associated with a sensor array 100 that measures physicaland/or chemical parameters in the microwells that provide informationabout the status of a reaction taking place therein; or in the case ofempty wells, information about the physical and/or chemical environmentin the flow cell. Each microwell may have a sensor for detecting ananalyte or reaction property of interest. In this particular embodiment,the microwell array is integrated with the sensor array as a singlechip, as explained more fully below. A flow cell can have a variety ofdesigns for controlling the path and flow rate of reagents over themicrowell array. In some embodiments, a flow cell is a microfluidicsdevice, which may be fabricated with micromachining techniques orprecision molding to include additional fluidic passages, chambers, andso on. This particular apparatus has an array controller 126 whichreceives information from sensor array 100 and reference electrode 108via communication line 126. A user interface 128 provides an interfacethrough which a user may interact with the apparatus.

FIG. 6 is an expanded and cross-sectional view of flow cell 200 showinga portion of a flow chamber 206 with reagent flow 208 moving across thesurface of microwell array 202 over the open ends of the microwells.Preferably, microwell array 202 and sensor array 205 together form anintegrated unit forming a bottom wall or floor of flow cell 200. In oneembodiment, reference electrode 204 is fluidly connected to flow chamber206. A microwell 201 and sensor 214 are shown in an expanded view.Microwell 201 is formed in the bulk material 210 by any conventionalmicrofabrication technique. The volume, shape, aspect ratio (such asbase width-to-well depth ratio), and other dimensional characteristicsof the microwells are design choices that depend on a particularapplication, including the nature of the reaction taking place, as wellas the reagents, byproducts, and labeling techniques (if any) that areemployed. Sensor 214 is a chemFET with a floating gate 218 having sensorplate 220 separated from the microwell interior by passivation layer216. Sensor 214 is predominantly responsive to (and generates an outputsignal related to) the amount of charge 224 present on the passivationlayer 216 opposite of sensor plate 220. Changes in charge 224 causechanges in the current between source 221 and drain 222 of the FET,which may be used directly to provide a current-based output signal orindirectly with additional circuitry to provide a voltage-based outputsignal. Reactants, wash solutions, and other reagents move intomicrowells from flow chamber 206 primarily by diffusion 240.

In another embodiment of the present teaching, a fluidics controller(e.g., fluidics controller 118 in FIG. 5) is programmed to cause theflow of dNTP reagents in the manner described above. Another embodimentof the present teachings includes a computer-readable storage mediumhaving executable instructions for performing the sequencing methodsdescribed above. The storage medium may be any type of computer-readablemedium (i.e., one capable of being read by a computer), includingnon-transitory storage mediums such as magnetic or optical tape or disks(e.g., hard disk or CD-ROM), solid state volatile or non-volatilememory, including random access memory (RAM), read-only memory (ROM),electronically programmable memory (EPROM or EEPROM), or flash memory.The term “non-transitory computer-readable storage medium” encompassesall computer-readable storage media, but excludes a transitory,propagating signal. As explained above, the instructions on thecomputer-readable storage medium may control the operation of a fluidicscontroller or sequencing apparatus of the present teachings.

Terminology

Unless otherwise specifically designated herein, terms and symbols ofnucleic acid chemistry, biochemistry, genetics, and molecular biologyused herein follow those of standard treatises and texts in the field.See, for example, Kornberg and Baker, DNA REPLICATION, 2nd ed. (W.H.Freeman, New York, 1992); Lehninger, BIOCHEMISTRY, 2nd ed. (WorthPublishers, New York, 1975); Strachan and Read, HUMAN MOLECULARGENETICS, 2nd ed. (Wiley-Less, New York, 1999).

“Microwell,” which is used interchangeably with “reaction chamber,” mayrefer to a special case of a “reaction confinement region” or “reactionarea,” that is, a physical or chemical attribute of a substrate thatpermit the localization of a reaction of interest. Reaction confinementregions may be a discrete region of a surface of a substrate thatspecifically binds an analyte of interest, such as a discrete regionwith oligonucleotides or antibodies covalently linked to such surface.Reaction confinement regions may be configured or associated withstructural attributes such as hollows or wells having defined shapes andvolumes which are manufactured into a substrate. These latter types ofreaction confinement regions may be microwells or reaction chambers, andmay be fabricated using conventional microfabrication techniques, suchas those described in the following references: Doering and Nishi(eds.), HANDBOOK OF SEMICONDUCTOR MANUFACTURING TECHNOLOGY, 2nd ed. (CRCPress, 2007); Saliterman, FUNDAMENTALS OF BIOMEMS AND MEDICALMICRODEVICES (SPIE Publications, 2006); Elwenspoek et al, SILICONMICROMACHINING (Cambridge University Press, 2004); and the like. Variousconfigurations (e.g., spacing, shape and volumes) of microwells orreaction chambers are disclosed in Rothberg et al, U.S. PatentPublication 2009/0127589 and No. 2009/0026082; Rothberg et al, UK.Patent Application GB2461127; and Kim et al., U.S. Pat. No. 7,785,862,which are incorporated by reference. The microwells may have anysuitable shape, such as square, rectangular, or octagonal crosssections, and may be arranged as a rectilinear array on a surface.Microwells may also have hexagonal cross sections and be arranged as ahexagonal array, which permit a higher density of microwells per unitarea in comparison to rectilinear arrays. In some embodiments, thereaction chamber array comprises 10², 10³, 10⁴, 10⁵, 10⁶ or 10⁷ reactionchambers.

An array is a planar arrangement of elements such as sensors or wells. Aone dimensional array is an array having one column (or row) of elementsin the first dimension and a plurality of columns (or rows) in thesecond dimension. The number of columns (or rows) in the first andsecond dimensions may or may not be the same. Preferably, the array usedin the present teachings comprises at least 100,000 chambers.Preferably, each reaction chamber has a horizontal width and a verticaldepth that has an aspect ratio of about 1:1 or less. Preferably, thepitch between the reaction chambers is no more than about 10 microns.Briefly, in one embodiment microwell arrays may be fabricated asfollows. After the semiconductor structures of a sensor array areformed, the microwell structure is applied to such structure on thesemiconductor die. That is, the microwell structure can be formed righton the die or it may be formed separately and then mounted onto the die,either approach being acceptable. To form the microwell structure on thedie, various processes may be used. For example, the entire die may bespin-coated with a negative photoresist such as Microchem's SU-82015 ora positive resist/polyimide such as HD Microsystems HD8820, to thedesired height of the microwells. The desired height of the wells (e.g.,about 3-12 μm in the example of one pixel per well, though not solimited as a general matter) in the photoresist layer(s) can be achievedby spinning the appropriate resist at predetermined rates (which can befound by reference to the literature and manufacturer specifications, orempirically), in one or more layers. Well height typically may beselected in correspondence with the lateral dimension of the sensorpixel. For example, the wells may have a nominal 1:1 to 1.5:1 aspectratio, height:width or diameter. Alternatively, multiple layers ofdifferent photoresists may be applied or another form of dielectricmaterial may be deposited. Various types of chemical vapor depositionmay also be used to build up a layer of materials suitable for microwellformation therein. In one embodiment, microwells are formed in a layerof tetra-methyl-ortho-silicate (TEOS).

The present teachings encompass an apparatus comprising at least onetwo-dimensional array of reaction chambers, wherein each reactionchamber is coupled to a chemically-sensitive field effect transistor(“chemFET”) and each reaction chamber is no greater than 10 μm³ (i.e., 1pL) in volume. Preferably, each reaction chamber is no greater than 0.34pL, and more preferably no greater than 0.096 pL or even 0.012 pL involume. A reaction chamber can optionally be 2², 3², 4², 5², 6², 7², 8²,9², or 10² square microns in cross-sectional area at the top.Preferably, the array has at least 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸,10⁹, or more reaction chambers. The reaction chambers may becapacitively coupled to the chemFETs.

“Polynucleotide” or “oligonucleotide” are used interchangeably and referto a linear polymer of nucleotide monomers. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Such monomers and their internucleosidic linkagesmay be naturally occurring or may be analogs thereof, e.g., naturallyoccurring or non-naturally occurring analogs. Non-naturally occurringanalogs may include PNAs, phosphorothioate internucleosidic linkages,bases containing linking groups permitting the attachment of labels,such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such asextension by a polymerase, ligation by a ligase, or the like, one ofordinary skill would understand that oligonucleotides or polynucleotidesin those instances would not contain certain analogs of internucleosidiclinkages, sugar moieties, or bases at any or some positions.

Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. Whenever a polynucleotide oroligonucleotide is represented by a sequence of letters (upper or lowercase), such as “ATGCCTG,” it will be understood that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, “I” denotes deoxyinosine, “U” denotes uridine, unlessotherwise indicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, HUMAN MOLECULAR GENETICS 2 (Wiley-Liss, New York,1999).

Usually polynucleotides comprise the four natural nucleosides (e.g.deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA ortheir ribose counterparts for RNA) linked by phosphodiester linkages;however, they may also comprise non-natural nucleotide analogs, e.g.including modified bases, sugars, or internucleosidic linkages. It isclear to those skilled in the art that where an enzyme has specificoligonucleotide or polynucleotide substrate requirements for activity,e.g. single stranded DNA, RNA/DNA duplex, or the like, then selection ofan appropriate composition for the oligonucleotide or polynucleotidesubstrates is well within the knowledge of one of ordinary skill,especially with guidance from treatises such as Sambrook et al,MOLECULAR CLONING, 2nd ed. (Cold Spring Harbor Laboratory, New York,1989), and like references.

“Primer” refers to an oligonucleotide, either natural or synthetic thatis capable, upon forming a duplex with a polynucleotide template, ofacting as a point of initiation of polynucleic acid synthesis and beingextended from its 3′ end along the template so that an extended duplexis formed. Extension of a primer may be carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Primers may be extended by a DNA polymerase.Primers may have a length in the range of from 14 to 40 nucleotides, orin the range of from 18 to 36 nucleotides. Primers are employed in avariety of amplification reactions, for example, linear amplificationreactions using a single primer, or polymerase chain reactions,employing two or more primers. Guidance for selecting the lengths andsequences of primers for particular applications is well known to thoseof ordinary skill in the art, as evidenced by the following referencesthat are incorporated by reference: Dieffenbach (ed.), PCR PRIMER: ALABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, New York, 2003).

While the present teachings has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present teachings. The present teachings are applicableto a variety of sensor implementations and other subject matter, inaddition to those discussed above.

1. A method for sequencing a template polynucleotide strand comprising:(a) disposing a plurality of template polynucleotide strands into aplurality of reaction chambers, each reaction chamber comprising atemplate polynucleotide strand having a sequencing primer hybridizedthereto and a polymerase operably bound thereto; (b) introducing a knownnucleoside triphosphates into each reaction chamber according to apredetermined ordering of dNTP flows; (c) detecting sequentialincorporation at the 3′ end of the sequencing primer of one or morenucleoside triphosphates if the known nucleoside triphosphate iscomplementary to corresponding nucleotides in the template nucleic acid;(d) washing away unincorporated nucleoside triphosphates from thereaction chamber; and (e) repeating steps (b) through (d) until thepolynucleotide strand is sequenced; wherein the predetermined orderingof dNTP flows is defined by (i) a flow of one kind is always followed bya flow of a different kind; and (iii) at least one flow of each kind isfollowed by a flow of the same kind after a single intervening flow of adifferent kind.
 2. The method of claim 1, wherein the number of flows ofeach kind in the plurality is the same.
 3. A method of sequencing apolynucleotide strand, comprising: providing the polynucleotide strandwith a primer annealed thereto and a polymerase operably bound to thepolynucleotide strand; successively exposing the polynucleotide strandto the flow of four different dNTPs according to a first predeterminedordering; and successively exposing the polynucleotide strand to theflow of four different dNTPs according to a second predeterminedordering, wherein the second predetermined ordering is different fromthe first predetermined ordering.
 4. The method of claim 3, wherein thesecond predetermined ordering comprises an alternate ordering which isnot a continuous repeat of the first predetermined ordering of the fourdifferent dNTPs.
 5. The method of claim 3, further comprisingsuccessively exposing the polynucleotide strand to the flow of fourdifferent dNTPs according to a third predetermined ordering, wherein thethird predetermined ordering comprises an alternate ordering which isnot a continuous repeat the of the first predetermined ordering of thefour different dNTPs or of the second predetermined ordering of the fourdifferent dNTPs.
 6. The method of claim 3, further comprisingsuccessively exposing the polynucleotide strand to the flow of fourdifferent dNTPs according to the first predetermined ordering aftersuccessively exposing the polynucleotide strand to the flow of fourdifferent dNTPs according to the second predetermined ordering.